Dental caries is a multi-faceted disease caused by interactions of cariogenic bacteria present in oral biofilms with salivary components and dietary carbohydrates. Despite significant advances in our understanding of the etiology and pathogenesis of this disease, dental caries remains the most prevalent and costly biofilm-associated infection worldwide. Among the hundreds of bacterial species residing in the oral biofilm, Streptococcus mutans has long been known as a primary cariogenic agent by its ability to drive the ecological transition of the biofilm population from non-cariogenic to cariogenic. The capacities to synthesize a thick extracellular polysaccharide matrix from sucrose, to generate copious amounts of lactic acid through carbohydrate fermentation, and to rapidly adapt to environmental stresses are the key virulence attributes of S. mutans. Although acidogenicity and aciduricity are well-established stress factors involved in virulence, the ability to cope with endogenous and exogenous reactive oxygen species is also viewed as an important attribute in the pathophysiology of S. mutans. This competing renewal has been set forth to study, at the molecular and physiologic levels, the Spx oxidative stress regulators of S. mutans. The Spx protein is highly conserved among Firmicutes and elegant studies with the Gram-positive paradigm Bacillus subtilis have shown that it exerts positive control over oxidative stress genes through direct interactions with the RNA polymerase and the promoter DNA region. Our work in the previous funding cycle identified two bona fide Spx paralogs (SpxA1 and SpxA2) in the genome of S. mutans, and showed that stress tolerances and virulence are significantly impaired in strains lacking one or both spx genes. Following our initial study, evidence of two Spx regulatory systems emerged in other bacteria and the importance of Spx regulation to virulence has been expanded to other streptococcal species. Although the spx gene has been well characterized in B. subtilis, the interplay among two Spx paralogs and the scope of Spx regulation in pathogenic organisms such as S. mutans are not well understood. The goals of this project are to understand the hierarchical relationship of the two Spx paralogs and uncover novel, Spx regulated, antioxidant strategies in S. mutans. To accomplish these goals, we propose three specific aims. In Aim 1, we will unravel the regulatory network controlling cellular abundance of the Spx proteins. In the second aim, we will determine, at the molecular level, the regulatory capacities of each Spx protein in relation to oxidative stress gene activation. In the third aim, we will take advantage of the prominent role of SpxA1 in activation of oxidative stress responses to uncover how S. mutans respond to peroxide stress at the metabolic level and to identify novel antioxidant pathways. The successful completion of this project will provide new leads on bacterial processes that can be exploited for new therapeutic and preventive strategies.